Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the U.S. Only a subset of patients initially responds to the current chemotherapeutic agent gemcitabine. Traditional cytotoxic agents are ineffective at controlling the signaling pathways that ultimately drive the disease progression. Hence, there is an urgent need to target signaling pathways specifically amplified during disease progression. An important theme that has emerged from our preliminary studies is that afatinib, an irreversible inhibitor of all ErbB family members, would be an alternative strategy for the combined approach of targeting all EGFR family members. Through this proposal we seek to develop an effective therapy for PC by combining the afatinib and the cytotoxic agent gemcitabine to provide broad spectrum molecular (EGFR family proteins and also mucins) targeted therapeutic protocol with enhanced antitumor efficacy and control of chemoresistance. Based on previous studies and our preliminary observations we hypothesize that afatinib, a pan-EGFR family inhibitor will enhance the efficacy of gemcitabine by modulating/downregulation EGFR family proteins along with mucins, augmenting tumor microenvironment and targeting cancer stem cells. We have proposed three specific aims to test this hypothesis. The first aim is to evaluate the functional effect of afatinib, alone and in combination with gemcitabine in PC cells, functiona significance and intrinsic resistance by using the combinational approach in PC cells. In this aim we will explore the mechanism of action of afatinib in augmenting mucin-mediated therapeutic resistance in PC cells. We will also predict the sensitivity of the combination of gemcitabine with afatinib in overcoming intrinsic resistance in vitro in PC cells. The second aim is focused at exploring the efficacy of the combination of gemcitabine with afatinib in genetically engineered mouse model of PC. The third aim is to investigate the potential impact of pan-EGFR Inhibitor in overcoming intrinsic resistance imposed by pancreatic cancer stem cells (PCSC) and tumor microenvironment. In this aim, we will analyze the effect of combination therapy on pancreatic cancer stem cells and tumor microenvironment to overcome the chemotherapeutic resistance. The significance of the present proposal is to understand the central mechanism(s) of the afatinib-induced enhanced chemotherapeutic efficacy of gemcitabine in lethal PC. Altogether, the overall results of this proposal would be enormously promising for initiating clinical trials consisting of a combination of EGFR family member specific inhibitor afatinib with gemcitabine as a therapeutic tool in PC patients. The outcome of the proposed studies will have high impact on thousands of pancreatic cancer patients currently receiving gemcitabine as a first line chemotherapy.